Pantoea agglomerans ALC and application thereof in preparation of aluminum-toxic soil plant growth promoting bacterial fertilizer

Abstract

The invention discloses pantoea agglomerans ALC and application thereof in preparation of a plant growth-promoting bacterial fertilizer for aluminum-toxic soil, and belongs to the field of soil remediation, the pantoea agglomerans ALC is preserved in China General Microbiological Culture Collection Center on July 15, 2024, and the preservation number is CGMCC No.31309. The invention further discloses a preparation method of the pantoea agglomerans ALC and application of the pantoea agglomerans ALC in preparation of a plant growth-promoting bacterial fertilizer for aluminum-toxic soil. On one hand, citric acid is generated through the pantoea agglomerans ALC to chelate active aluminum, on the other hand, phosphate is generated through the pantoea agglomerans ALC to reduce the content of the active aluminum in the soil, and finally the content of the active aluminum in the aluminum-toxic soil is reduced, so that damage to plants and crops is reduced. The plant growth-promoting bacterial fertilizer for the aluminum-toxic soil, prepared by the invention, can be used for restoring heavy metal contaminated soil and also can be used for reducing stress of heavy metals on plants, promoting plant growth and increasing soil fertility, and a plurality of microorganisms have a synergistic effect, so that the restoration efficiency of the heavy metal soil is further improved through the synergistic effect.

Description

Technical Field

[0001] The invention belongs to the technical field of soil remediation, and specifically relates to a strain of Pantoea agglomerans ALC and application thereof in preparing a plant growth-promoting bacterial fertilizer for aluminum-toxic soil. Background Art

[0002] Active aluminum has adverse effects on plant growth and soil microbial activity. When soil pH < 5, aluminum in the soil is reduced to A1 3+ It is released into the soil liquid in the form of aluminum and absorbed by the plant roots. In the soil environment poisoned by heavy metal aluminum, it will affect the germination of seeds. Seeds are very fragile in the germination stage and are easily damaged by adverse stress. The aluminum stress environment has a high ion toxicity, which can destroy many physiological and biochemical reaction processes inside the seeds and inhibit seed germination. Aluminum stress will also affect the growth and development of crops. The most important feature of plants poisoned by aluminum is the inhibition of root elongation, the impact of water and nutrient absorption, and thus the growth and development of plants. And most plants will show symptoms of poisoning under very low concentrations (several μM) of aluminum stress. When plants are harmed by aluminum, the elongation of the root main axis is inhibited, making the plant root system become thick and short, the color turns brown, the root crown falls off, the branches are reduced, the germination of adventitious roots is inhibited, and the root tip structure is destroyed. A large amount of Al 3+ When present, it can even cause root necrosis. Aluminum toxicity affects the absorption and metabolism of plant nutrients, causing the aboveground part of the plant to show symptoms similar to those of nutrient deficiency. After aluminum enters the plant body, the young leaves become smaller, the leaves turn yellow and the leaf edges curl, and the top growth point of the plant wilts, similar to the symptoms of calcium or phosphorus deficiency. Under the influence of high aluminum toxicity stress, plant growth and development are inhibited or even the plant dies. In addition, after aluminum enters plant cells, it will destroy the structure and function of the cell membrane, change the fluidity and permeability of the membrane, affect the normal operation of membrane transfer proteins, and interfere with cell signal regulation and material transport processes. Aluminum toxicity can also destroy the hormone balance in the plant body and cause changes in cytokinins. Therefore, the toxicity of aluminum to plants in acidic soils has received widespread attention. Biological improvement methods have attracted much attention due to their high economic benefits and green environmental protection. Using the characteristics of microorganisms to produce citric acid to improve acidic aluminum-toxic soils and reduce the impact of aluminum stress on plants has great research prospects.

[0003] Among the tolerance mechanisms evolved by plants to cope with aluminum stress, root organic acid secretion is considered to be the most important aluminum exclusion pathway. A large number of studies have shown that under aluminum stress, plants promote root secretion of low-molecular organic acid compounds, which chelate with aluminum in the rhizosphere to form non-toxic organic acid-aluminum compounds, preventing aluminum from entering root cells. The aluminum tolerance mechanism, the extracellular chelation of aluminum ions and organic acids, is determined in aluminum-tolerant higher plants, but microorganisms known as the second genome of plants also have a variety of anti-aluminum toxicity characteristics. Some microorganisms secrete organic acids to convert Al into aluminum. 3+Chelate outside the cell, thereby reducing the toxicity of Al. For example, microorganisms can produce citric acid and Al 3+ Chelated with Al 3+ -citric acid (1:1 molar ratio) to reduce aluminum toxicity in the soil.

[0004] Phosphorus is one of the essential nutrients for plant growth and development. It plays an important role in plant energy transfer, signal transduction, photosynthesis, cell division and enlargement, and macromolecular biosynthesis. Although the soil is rich in phosphorus, most of it is inorganic phosphorus. The effective phosphorus content in the soil that can be absorbed and utilized by plants accounts for only 0.1% of the total phosphorus. Therefore, in agricultural production, the application of phosphorus fertilizer is usually increased to meet the crop's demand for phosphorus. However, after phosphorus fertilizer is applied to the soil, it is easy to react with Ca in the soil. 2+ , Fe 3+ 、Al 3+ Plasma combines to form insoluble phosphates. The abuse of chemical phosphate fertilizers not only increases the cost of agricultural production, but also destroys the diversity of soil microorganisms, leading to weakened soil fertility and reduced crop yields, and exacerbating problems such as eutrophication of water bodies and deterioration of the water environment caused by soil phosphorus loss. There are a large number of phosphate-solubilizing microorganisms in the environment that can convert phosphorus that is difficult for plants to use into absorbable forms, including bacteria, fungi, actinomycetes, and cyanobacteria. Among them, phosphate-solubilizing bacteria are the most numerous and the most widely used. Applying phosphate-solubilizing bacteria to the soil can not only increase the amount of phosphorus absorbed by plants and increase crop yields, but also greatly improve the utilization rate of phosphate fertilizers and reduce the risk of environmental pollution caused by them.

[0005] Potassium is one of the important nutrients required for plant growth and development. Potassium can promote photosynthesis in plants, thereby increasing the assimilation rate of CO2, promoting the transportation of photosynthesis products, participating in the synthesis and transmission of plant growth hormones, activating enzyme activity, and thus promoting plant growth. It participates in regulating the ion balance of plants, maintaining cell osmotic regulation and water balance, promoting photosynthesis and protein synthesis in plants, and improving plant stress resistance and yield. A large number of agricultural production practices have proved that the lack of potassium may lead to problems such as poor plant development and leaf dryness. Therefore, microorganisms with potassium-solubilizing function have great research significance.

[0006] Self-generated nitrogen-fixing bacteria do not coexist with higher plants, but can survive independently in the soil. They use organic matter in the soil or organic matter secreted by the roots as a carbon source to convert nitrogen in the atmosphere into nitrogen that can be absorbed and utilized by plants, providing a continuous supply for plants to enjoy, or directly use inorganic nitrogen compounds in the soil to increase nitrogen in the soil. Nitrogen plays a vital role in plants. It is the main component of proteins, nucleic acids, and phospholipids, and occupies a special position in life activities. It is also a component of many enzymes, coenzymes, and certain plant hormones such as auxin and cytokinin, which regulates the life activities of plants. It is also a component of chlorophyll and is closely related to photosynthesis. Therefore, self-generated nitrogen-fixing microorganisms have certain research value.

[0007] IAA is the most abundant auxin-like substance produced by microorganisms. As a plant hormone that plays an important regulatory role in plants, it can promote plant cell division and elongation, promote stomatal opening, stimulate growth and reduce root elongation, increase the activity of plant antioxidant enzymes, enhance plant disease resistance, etc., thereby achieving the purpose of promoting plant growth.

[0008] Iron-producing bacteria not only play a very important role in the growth and development of plants, but also have a great impact on environmental pollution remediation. When the soil is in a low-iron stress environment, bacteria can produce iron carriers to form iron chelates, which can effectively absorb the low-solubility iron in the environment to promote plant growth, and can react with heavy metal ions to chelate. Iron carriers are one of the trace nutrients necessary for the normal growth of crops. Iron is not only involved in physiological processes such as plant chlorophyll synthesis, respiration, and redox reactions, but is also an important auxiliary factor for many functional proteins. And iron carriers have the advantages of low cost, high efficiency, and no environmental pollution in repairing heavy metal pollution. Bacteria have the advantages of wider sources, more types, and faster growth, and have good application prospects in the production of iron carriers.

[0009] The formation of acid-aluminum soil is, on the one hand, due to the acidification of rainwater, and aluminum is released (dissolved) from natural reservoirs. On the other hand, industrialization and modernization have jointly led to the excessive accumulation of heavy metals. In this context, improving acid-aluminum soil and reducing the impact of aluminum stress on plants have become urgent issues to be addressed. Low pH is the main cause of aluminum (Al) production and is considered to be an important limiting factor for plant growth, especially when pH <5. A large number of studies have shown that using the characteristics of microorganisms to produce phosphate and citric acid to improve acid-aluminum soil has great research prospects.

[0010] At present, there are many bacterial fertilizers for improving saline-alkali land on the market, but there are few bacterial agents for improving and alleviating acidic aluminum toxicity soil. The traditional method for improving acidic soil is mainly chemical improvement, including: (1) applying lime to precipitate exchangeable aluminum into insoluble light-based aluminum, increasing aluminum saturation to alleviate the inhibition of crop growth and increase soil pH. However, the application of lime has the following problems: in terms of application method, the usual application of lime on the soil surface cannot effectively neutralize the soil acidity of the central soil layer, and the large amount used is also too costly. (2) Applying calcium magnesium phosphate fertilizer, calcium and magnesium are closely related to aluminum toxicity, and its alkalinity can neutralize the acidity of acid-aluminum soil, but excessive application can easily lead to resource waste and environmental pollution. (3) Applying organic matter to acidic soil, organic matter can effectively alleviate aluminum toxicity and improve the utilization rate of phosphorus, but the operation is cumbersome.

[0011] The above three acid soil conditioners can improve acid soil to a certain extent, increase soil pH, reduce the aluminum content in the soil, and thus improve soil acidity. However, these three conditioners have serious side effects, and they are frequently applied, with high costs and cumbersome operations, which can easily lead to waste of resources and environmental pollution, and there is a serious risk of soil heavy metal pollution. Summary of the invention

[0012] In order to solve the problems of existing acidic soil conditioners such as frequent application, high cost, complicated operation, easy waste of resources, environmental pollution and serious heavy metal pollution in the soil, the present invention provides a Pantoea agglomerans ALC and its application in preparing aluminum-toxic soil plant growth-promoting bacterial fertilizer.

[0013] The purpose of the present invention is to improve aluminum-toxic soil by using microbial resources and promote plant growth. Based on this, the present invention screens out acid- and aluminum-resistant Pantoea agglomerans ALC from acid-aluminum-contaminated soil samples, and has the functions of producing citric acid, IAA, siderophore and phosphate dissolving, and prepares it into bacterial fertilizer and applies it to aluminum-toxic soil, and simultaneously selects soybean as the test plant for potted plant experiment. The purpose of improving aluminum-toxic soil and reducing aluminum stress on plants is achieved by applying a composite bacterial fertilizer composed of Pantoea agglomerans ALC and fulvic acid.

[0014] The present invention can chelate active aluminum by producing citric acid through Pantoea agglomerans ALC on the one hand, and reduce the content of active aluminum in the soil by producing phosphate through Pantoea agglomerans ALC on the other hand, thereby ultimately reducing the content of active aluminum in aluminum-toxic soil, thereby reducing damage to plants and crops.

[0015] The aluminum-toxic soil plant growth-promoting bacterial fertilizer prepared by the present invention can not only repair heavy metal-contaminated soil, but also reduce the stress of heavy metals on plants, promote plant growth, and increase soil fertility. There is a synergistic effect between the various microorganisms, and the efficiency of heavy metal soil remediation is further improved through this synergistic effect.

[0016] The technical solution adopted by the present invention to solve the technical problem is as follows:

[0017] In a first aspect, the present invention provides a strain of Pantoea agglomerans ALC, which has been deposited in the General Microbiology Center of China National Microbiological Culture Collection Administration on July 15, 2024, with the deposit number: CGMCC No.31309.

[0018] In a second aspect, the present invention provides a use of Pantoea agglomerans ALC in preparing a bacterial fertilizer for promoting plant growth in aluminum-toxic soil.

[0019] As a preferred embodiment, the functions of the Pantoea agglomerans ALC are:

[0020] (1) Phosphate solubilization;

[0021] (2) potassium release;

[0022] (3) Nitrogen fixation;

[0023] (4) Producing citric acid;

[0024] (5)Production of IAA;

[0025] (6) Producing iron carriers.

[0026] In a third aspect, the present invention provides an aluminum-toxic soil plant growth-promoting bacterial fertilizer, comprising a strain of Pantoea agglomerans ALC described in the first aspect.

[0027] As a preferred embodiment, the aluminum-toxic soil plant growth-promoting bacterial fertilizer provided by the present invention also includes fulvic acid.

[0028] As a preferred embodiment, after Pantoea agglomerans ALC is fermented and cultured, fulvic acid and the fermentation liquid of Pantoea agglomerans ALC are mixed evenly according to the OD600 value of the fermentation liquid of Pantoea agglomerans ALC and the colony count of the OD600 value so that the effective viable bacteria count in the fulvic acid is greater than 2.0×10 8cfu / g.

[0029] In a fourth aspect, the present invention provides a method for preparing an aluminum-toxic soil plant growth-promoting bacterial fertilizer, which specifically comprises the following steps:

[0030] Pantoea agglomerans ALC was inoculated into LB liquid medium for fermentation culture; the fermentation liquid of Pantoea agglomerans ALC was mixed with fulvic acid evenly, and the effective viable bacteria count of fulvic acid was adjusted to >2.0×10 8 cfu / g, prepared into solid bacterial fertilizer.

[0031] As a preferred embodiment, the fermentation culture conditions are: 30°C, 180rpm, culturing for 2 days.

[0032] As a preferred embodiment, the LB liquid culture medium contains 5 g / L yeast extract, 10 g / L tryptone, 10 g / L sodium chloride, and a pH of 4.5; the LB solid culture medium contains 5 g / L yeast extract, 10 g / L tryptone, 10 g / L sodium chloride, and 40 g / L agar, and a pH of 4.5.

[0033] In a fifth aspect, the present invention provides an application of aluminum-toxic soil plant growth-promoting bacterial fertilizer in aluminum-toxic soil remediation and plant growth promotion.

[0034] The present invention has the following beneficial effects:

[0035] The Pantoea agglomerans ALC screened by the present invention has a high citric acid production content, a strong ability of the strain to dissolve phosphorus, is environmentally friendly and efficient, can produce IAA and siderophores while reducing pH and aluminum toxicity, can promote plant growth, and also has the ability to dissolve phosphorus, potassium and fix nitrogen to increase soil fertility.

[0036] The Pantoea agglomerans ALC screened by the present invention and humic acid are applied together to soil containing aluminum poisoning. Through a potted soybean experiment, it is found that the aluminum-toxic soil plant growth-promoting bacterial fertilizer prepared by the present invention can effectively improve the germination rate of soybean seeds, has a growth-promoting effect on the roots and stems of soybeans, improves the aluminum resistance of soybeans, and improves the content of available nutrients such as nitrogen, phosphorus, potassium and organic matter in the aluminum-toxic soil, reduces the harmful effects of aluminum on plants, and realizes a virtuous cycle of the soil ecological environment, which is of great significance to agriculture. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 This is the result of the citric acid production ability test of strain ALC.

[0038] Figure 2This is the growth of strain ALC on potassium feldspar solid medium.

[0039] Figure 3 The growth of strain ALC on tricalcium phosphate solid medium.

[0040] Figure 4 This is the growth of strain ALC on Axubei solid culture medium.

[0041] Figure 5 This is the result of the IAA production capacity test of strain ALC.

[0042] Figure 6 This is the result of the determination of the iron carrier production ability of strain ALC.

[0043] Figure 7 The results of the strain ALC antagonism determination.

[0044] Figure 8 This is the effect of bacterial fertilizer treatment on soybean germination rate.

[0045] Fig. 9 The effect of bacterial fertilizer treatment on soybean root length and stem length. In the figure, A is the sterile agent (CK) control group, B is the fulvic acid treatment group, and C is the ALC bacterial agent treatment group.

[0046] Fig.10 The effect of bacterial fertilizer treatment on soybean root length.

[0047] Fig.11 This is the effect of bacterial fertilizer treatment on soybean stem length.

[0048] Fig.12 The effect of bacterial fertilizer treatment on soil nutrient index. In the figure, A: The effect of different bacterial fertilizer treatments on available potassium. B: The effect of different bacterial fertilizer treatments on alkaline nitrogen; C: The effect of different bacterial fertilizer treatments on the change of available phosphorus; D: The effect of different bacterial fertilizer treatments on organic matter; E: The effect of different bacterial fertilizer treatments on pH.

[0049] Fig.13 Soybean root tips stained with hematoxylin.

[0050] Fig.14 The soybean root tip was stained with mulberry pigment. DETAILED DESCRIPTION

[0051] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

[0052] Example 1 Screening, identification and preservation of strain ALC

[0053] (1) Initial bacterial screening

[0054] Aluminum-contaminated soil samples were collected from Shiwudaogou, Linjiang City, Jilin Province. 5 g of soil samples were placed in a triangular flask filled with 50 mL of sterile water to make soil dilution. The solution was shaken 2-3 times and allowed to settle. 5 mL of the upper supernatant was inoculated into a pH 4.5, Al 3+ 100 mL LB liquid medium with a final concentration of 1 mM; culture at 37°C, 180 rpm for 2-3 days; take 1 mL and dilute it 10-fold to get 10 -1 , 10 -2 , 10 -3 , 10 -4 , 10 -5 Bacterial suspension, take 100 μL of 10 -3 , 10 -4 , 10 -5 The bacterial suspension was spread on an aluminum-resistant screening solid medium (5 g / L yeast extract, 10 g / L tryptone, 15 g / L sodium chloride, 40 g / L agar, pH 4.5) with a pH of 4.5, and cultured at 37°C for 1-2 days; single colonies with good growth and different morphology were selected and placed in LB liquid medium (5 g / L yeast extract, 10 g / L tryptone, 10 g / L sodium chloride, pH 4.5) for separation and purification to obtain strain ALC; finally, the strain was cultured in LB liquid medium until OD600 was 0.8, and then mixed with 40% glycerol at a ratio of 1:1 (v / v), and stored in a -80°C refrigerator for later use.

[0055] (2) Determination of citric acid production capacity

[0056] After activating the initially screened strain ALC using LB liquid culture medium, the strain ALC was disrupted by ultrasonic wave in an ice bath (power 300w, ultrasonic wave 3 seconds, interval 7 seconds, total time 3min), centrifuged at 12000g, 4℃ for 10min, and the supernatant was taken for later use; the citric acid content was determined using a citric acid content determination kit (Suzhou Grace Biotechnology Co., Ltd.) to explore the strongest citric acid production ability of strain ALC.

[0057] The results are as follows Figure 1 As shown, the citric acid production capacity of strain ALC was determined using a citric acid content determination kit, and the citric acid content was 898.42 mg / L, indicating that strain ALC had a strong ability to produce citric acid.

[0058] (3) Acid resistance test

[0059] After activating the strain ALC in the initial screening using LB liquid medium, the OD600 of the bacterial suspension reached 0.8, and 100 μL of the bacterial suspension was inoculated into LB liquid medium with pH values ​​of 3.8, 4, 4.2, 4.4, and 4.6, respectively. Each gradient was repeated 3 times, and cultured at 37°C and 180 rpm for 1-2 days. Then 100 μL was taken and diluted 10-fold in sequence to obtain 10 -1 , 10 -2 , 10 -3 , 10 -4 , 10 -5 Bacterial suspension, take 100 μL of 10 -3 , 10 -4 , 10 -5 The bacterial suspension was spread on a culture medium with a pH of 4.5 (5 g / L yeast extract, 10 g / L tryptone, 10 g / L sodium chloride, 40 g / L agar, pH 4.5) and incubated upside down at 37°C for 1-2 days to explore the strongest acid resistance of strain ALC.

[0060] (4) Determination of aluminum resistance

[0061] After activating the strain ALC in the initial screening using LB liquid culture medium, the OD600 of the bacterial suspension reached 0.8. 100 μL of the bacterial suspension was added to LB liquid culture medium containing 1 mM, 2 mM, 3 mM, 4 mM, and 5 mM, respectively. After culturing for 2-3 days, the bacterial suspension was diluted to 10 using the gradient dilution method. -5 , draw 100 μL of 10 -5 The bacterial liquid was dropped in the middle of the LB liquid culture medium with a pH value of 4.5, spread evenly, and repeated 3 times for each gradient; the culture was inverted at 37°C for 1-2 days to explore the strongest aluminum resistance of strain ALC.

[0062] The results are shown in Table 1. The screened strain ALC has strong acid and aluminum resistance.

[0063] Table 1 Determination of acid and aluminum resistance of strain ALC

[0064]

[0065] The ability of strain ALC to chelate aluminum was determined by acid-base titration, and the result showed that 0.9544 mmol was chelated, indicating that strain ALC had a strong ability to chelate aluminum.

[0066] (5) Determination of potassium-dissolving ability

[0067] The strain ALC screened in the initial screening was inoculated into potassium feldspar solid culture medium, and the bacteria whose colonies turned orange were the strain ALC with potassium-solubilizing function ( Figure 2). Among them, the potassium feldspar solid culture medium is: sucrose 5g / L, glucose 5g / L, ammonium sulfate 0.5g / L, yeast powder 0.5g / L, magnesium sulfate 0.3g / L, disodium hydrogen phosphate 2g / L, ferrous sulfate 0.03g / L, manganese sulfate 0.03g / L, potassium feldspar 2g / L, bromothymol blue 0.1g / L, agar 15g / L.

[0068] The potassium-solubilizing capacity of strain ALC was determined by alkaline potassium persulfate digestion and UV spectrophotometry, and the potassium-solubilizing rate was 32.98%.

[0069] (6) Determination of phosphate solubilization capacity

[0070] The strain ALC screened in the initial screening was inoculated into tricalcium phosphate solid culture medium, and the bacteria with transparent circles were observed to be the strain ALC with phosphate solubilization function ( Figure 3 ). Among them, the tricalcium phosphate solid culture medium is: glucose 10g / L, potassium chloride 0.3g / L, ammonium sulfate 0.5g / L, magnesium sulfate heptahydrate 0.3g / L, sodium chloride 0.3g / L, ferrous sulfate heptahydrate 0.03g / L, manganese sulfate heptahydrate 0.03g / L, tricalcium phosphate 5g / L, agar 15g / L.

[0071] like Figure 3 As shown, strain ALC can grow on tricalcium phosphate solid culture medium and has a phosphate-solubilizing zone. The amount of phosphate dissolved was determined by molybdenum antimony colorimetry, and the amount of phosphate dissolved was 86.96 μg / mL. Strain ALC has a strong ability to dissolve inorganic phosphorus.

[0072] (7) Determination of nitrogen fixation capacity

[0073] The ALC strains screened were inoculated into the Axubei solid culture medium, and the bacteria with nitrogen-fixing function were observed to have colony growth ( Figure 4 ). Among them, the Axubei solid culture medium is: 0.2g / L potassium hydrogen phosphate, 0.2g / L magnesium sulfate heptahydrate, 0.1g / L calcium sulfate dihydrate, 0.2g / L sodium chloride, 5g / L calcium carbonate, 10g / L mannitol, and 18g / L agar.

[0074] The nitrogen fixation capacity of strain ALC was determined by flame spectrophotometer, and the nitrogen fixation capacity was 1.19 μg / mL.

[0075] (8) Determination of IAA production capacity

[0076] After activating the initially screened strain ALC using LB liquid culture medium, make the bacterial solution OD600 reach 0.8, add 500 μL of bacterial suspension onto a white ceramic plate, and add an equal volume of Salkowski colorimetric solution (1 mL 0.5 mol / L FeCl3 + 50 mL 35% HClO4). The white ceramic plate is left standing at room temperature in the dark for 30 minutes and then observed. The red color indicates that the bacteria can secrete indoleacetic acid.

[0077] The results are as follows Figure 5 As shown, the IAA production capacity of strain ALC was determined by Salkowski colorimetry, and the content was 62.23 mg / L.

[0078] (9) Determination of siderophore production capacity

[0079] The ALC strains screened were inoculated into CAS medium, and the colonies that turned pink were bacteria with iron-producing function ( Figure 6 ). Among them, the CAS medium is: hexadecylamine bromide 0.073g / L, chrome azurol s 0.065g / L, ferric chloride hexahydrate 2.703g / L, ammonium chloride 2.5g / L, potassium dihydrogen phosphate 0.75g / L, sodium chloride 1.25g / L, sodium dihydrogen phosphate 5.905g / L, disodium hydrogen phosphate 24.27g / L.

[0080] The CAS method was used to determine the siderophore production capacity of strain ALC, and the (A / Ar) value was 0.79, indicating that strain ALC had a strong ability to produce siderophores.

[0081] (10) Physiological and biochemical identification of strain ALC

[0082] After the above steps (1)-(9), strain ALC was screened and subjected to physiological and biochemical identification. + represents positive, - represents negative, and ⊕ represents acid and gas production. The results are as follows:

[0083]

[0084] ALC is a Gram-negative bacterium belonging to the genus Pantoea agglomerans and is named ALC. Its registration number in GenBank is OQ954790.

[0085] Further physiological and biochemical tests showed that the strain ALC was positive in methyl red test, acetylmethyl carbinol (VP) test, indole test, nitrate reduction test and catalase test; the hydrogen sulfide test and urease test were negative; the strain could not hydrolyze tyrosine or liquefy gelatin, but could hydrolyze starch. ALC could utilize citrate, and the oxidase test was negative. ALC could utilize glucose and produce acid but not gas; it could utilize sucrose to produce acid and gas; ALC could utilize fructose, trehalose, mannose and lactose to produce acid but not gas, but could not utilize maltose.

[0086] (11) Molecular identification of strain ALC

[0087] After the above steps (1)-(9), strain ALC was screened and molecularly identified. The 16SrDNA sequence homology analysis process and results are as follows:

[0088] The genomic DNA of strain ALC was extracted, and a primer pair consisting of 16sF and 16sR (27FAGAGTTTGATCMTGGCTCAG; 1492R GGTTACCTTGTTACGACTT) was used for PCR amplification, and then the obtained amplified product was sequenced, wherein the 16s rDNA sequence is shown in SEQ ID NO: 1. The 16s rDNA sequence was subjected to homology comparison analysis in the GenBank library using the BLAST program, and it was found that the homology between strain ALC and Pantoea agglomerans reached 99.8%. Therefore, according to the above identification results, the extracted strain ALC was identified as Pantoea agglomerans.

[0089] (11) Deposit of strain ALC

[0090] The Pantoea agglomerans ALC of the present invention has been deposited in the General Microbiological Center of the China Microorganism Culture Collection Administration, referred to as CGMCC, on July 15, 2024, with the address: No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing (Institute of Microbiology, Chinese Academy of Sciences), and the deposit number is: CGMCC No.31309.

[0091] In summary, the Pantoea agglomerans ALC screened and obtained in the present invention can be used as a strain for preparing bacterial fertilizer, and a bacterial fertilizer with the ability to solubilize phosphorus, potassium, fix nitrogen, produce citric acid, produce IAA, and produce siderophores can be obtained.

[0092] Example 2 Preparation of plant growth-promoting bacterial fertilizer in aluminum-toxic soil

[0093] (1) Strain activation

[0094] Pantoea agglomerans ALC was inoculated into 100 mL of LB liquid culture medium and cultured overnight at 30°C and 180 rpm in a shaking incubator to obtain a seed solution.

[0095] (2) Expanding training

[0096] The seed solution in step (1) was inoculated into LB liquid culture medium and cultured at 30°C and 180 rpm for 2 days. The OD value of the fermentation liquid was measured by a spectrophotometer at 600 nm. The fermentation liquid was centrifuged to obtain bacterial cells, which were suspended with sterile water to make the OD value consistent with 0.8. The bacterial suspension with an OD of 0.8 was diluted to 10 by gradient dilution method. -5 , spread 100 μL on LB solid culture medium with a pH of 4.5, and culture it upside down at 37°C for 1-2 days. Count the number of live bacteria on the culture dish, set up three replicates, and calculate the number of live bacteria contained in 1 μL, 1OD based on the OD value and the number of live bacteria in the culture dish.

[0097] (3) Preparation of plant growth-promoting bacterial fertilizer in aluminum-toxic soil

[0098] The bacterial fertilizer obtained in step (2) was mixed with fulvic acid to make the effective number of live bacteria in the fulvic acid > 2.0 × 10 8 cfu / g, and prepared into solid bacterial fertilizer, thereby obtaining a bacterial fertilizer suitable for reducing aluminum toxicity in the soil and promoting plant growth.

[0099] In the present invention, adding fulvic acid to the bacterial fertilizer can absorb nitrogen in the soil and chelate with phosphorus, which is beneficial to the absorption of phosphorus by plants and improves the utilization rate of potassium. It can also adjust the pH of the soil and increase sudden fertility, thereby promoting plant growth.

[0100] Example 3 Effects of different bacterial fertilizer treatments on soybean seed germination

[0101] (1) Effect of Pantoea agglomerata ALC suspension on soybean germination rate

[0102] Williams 82 soybean seeds were selected as experimental materials. The soybean seeds were soaked in 1% sodium hypochlorite for 10 minutes for disinfection, then washed with sterile water for 6 times to remove the residual sodium hypochlorite, and placed on a sterile clean bench for use. 10 soybean seeds were placed in a sterilized culture dish for aseptic culture. The control group (CK) and the experimental group were added with effective viable bacteria count> 2.0×10 8 cfu / g of Pantoea agglomerata ALC suspension and 30 μmol / L aluminum chloride solution and 50 μmol / L aluminum chloride solution of the corresponding volume of the bacterial suspension. The pot experiment design is shown in Table 2.

[0103] Table 2

[0104] Group Different treatment CK 3 mL sterile distilled water 1 3mL of Pantoea agglomerata ALC suspension 2 <![CDATA[3mL30μmol / LAlCl3]]> 3 <![CDATA[3mL50μmol / LAlCl3]]> 4 <![CDATA[3 mL成团泛菌ALC bacterial suspension containing 30 μmol / L AlCl3]]> 5 <![CDATA[3 mL成团泛菌ALC bacterial suspension containing 50 μmol / L AlCl3]]>

[0105] The results are as follows Figure 8 As shown, the first row is the untreated group, the second row is the 30μM AlCl3 treated group, and the third row is the 50μM AlCl3 treated group. The first column is the sterile treatment group, and the second column is the bacterial agent (ALC) treatment group. After 7 days of culture with the best composite bacterial agent, the number of soybean germinations increased and the growth was better. It proves that the bacterial agent alleviates the effects of aluminum stress on soybeans.

[0106] (2) Effect of bacterial fertilizer on soybean germination rate

[0107] A pot experiment was used to study the growth-promoting effect of bacterial fertilizer on soybean. In the pot experiment, 50 soybean seeds were planted in each pot, and all samples were cultured at room temperature for 7 days to evaluate the germination of the tested soybean seeds. All experiments were repeated 3 times. The pot experiment design is shown in Table 3. Each pot contained 1 kg of original acidic aluminum-toxic soil (200 mesh).

[0108] Table 3

[0109] Group Different treatment CK No fertilizer added FA Add 0.3g fulvic acid 1 Add 0.3 g of the bacterial fertilizer prepared in step (2) of Example 2 (containing only Pantoea agglomerans ALC) 2 Add 0.3g aluminum-toxic soil plant growth-promoting bacterial fertilizer (containing Pantoea agglomerans ALC + fulvic acid)

[0110] The results showed that the germination rate of soybean seeds in the CK group was 40%, and the germination rate of soybean seeds in the experimental group (Group 2) was 72.67%.

[0111] Example 4 Effects of different bacterial fertilizer treatments on soybean morphological indicators

[0112] Seven seeds were randomly selected and planted in the original acidic aluminum-poisoned soil (200 mesh). When the second triplicate leaf of the second node stage unfolded, the soybean roots were taken, rinsed with distilled water, placed on filter paper to absorb water, and the root tip length (cm) and stem length (cm) were counted. Plants to be tested were randomly selected in each treatment group and repeated 3 times.

[0113] The results are as follows Figure 9-11 As shown, the soybean plants in the CK group were smaller and had weaker root systems. The root length and diameter length of the experimental group (LJ-1) were both improved compared with the CK group, and the soybean growth state was the best. This shows that the aluminum-toxic soil plant growth-promoting bacterial fertilizer prepared by the present invention has a good growth-promoting effect on the growth of plant roots and stems.

[0114] Example 5 Effects of different bacterial fertilizer treatments on soil nutrient indicators

[0115] A pot experiment was conducted to study the effect of bacterial fertilizer on soil nutrient indexes. The pot experiment design and grouping are shown in Table 2 in Example 3.

[0116] For the determination of available potassium, alkaline-hydrolyzable nitrogen, available phosphorus, organic matter and pH in acidic aluminum poisoning soil, the available potassium was determined by neutral ammonium acetate solution extraction-flame photometry; the alkaline-hydrolyzable nitrogen was determined by diffusion method; the available phosphorus was determined by sodium bicarbonate extraction-spectrophotometry; the organic matter was determined by potassium dichromate oxidation-oil bath heating method; and the soil pH was determined using a pH meter.

[0117] The results are as follows Fig.12 As shown, the soil alkaline nitrogen, available phosphorus, available potassium and pH contents of the experimental group (LJ-1) were 323.29 mg / kg, 11.51 mg / kg, 115 mg / kg and 5.11, respectively, which were all higher than those of the CK group. The aluminum-toxic soil plant growth-promoting bacterial fertilizer prepared by the present invention can significantly increase the content of available potassium, alkaline nitrogen, available phosphorus and organic matter in acidic aluminum-toxic soil, and can achieve the purpose of improving the nutrient content of acidic aluminum-toxic soil and increasing the soil pH value.

[0118] Example 6 Effects of different bacterial fertilizer treatments on aluminum toxicity in soybean root tips

[0119] A pot experiment was conducted to study the effect of bacterial fertilizer on aluminum toxicity in soybean root tips. The design and grouping of the pot experiment are shown in Table 2 in Example 3.

[0120] The soybean root tips were stained with hematoxylin and mulberry pigment to test the effect of acidic aluminum-toxic soil on reducing aluminum toxicity in soybean root tips after applying bacterial fertilizer. When soybeans grew to the three-leaf stage, fresh soybean roots were randomly taken, cleaned with CaCl2 solution, placed on filter paper to absorb moisture, and stained with hematoxylin and mulberry pigment for 30 minutes. The above indicators reflect the effect of the aluminum-toxic soil plant growth-promoting bacterial fertilizer prepared by the present invention on alleviating aluminum toxicity in soybean root tips. The results are as follows Fig.13 and Fig.14 As shown in the figure, after the ALC treatment, the soybean root tip was stained with hematoxylin and morin using an inverted microscope and a fluorescent microscope to examine the effect of aluminum stress on soybean. Fig.13 , Fig.14 As shown, the color of the groups B and D treated with bacterial agents was significantly lighter than that of the groups A and C treated without bacterial agents, proving that the aluminum content in the root tip decreased after the application of the composite bacterial agent, further confirming that the exchangeable aluminum content in the soil decreased after the bacterial agent treatment.

[0121] The aluminum-toxic soil plant growth-promoting bacterial fertilizer prepared by the present invention reduces the stress hazard of aluminum in aluminum-toxic soil by producing citric acid and combining with exchangeable aluminum in the soil. The bacterial fertilizer applied to aluminum-toxic soil can not only effectively reduce the exchangeable aluminum content in the soil, but also significantly increase the germination rate of soybean seeds and promote the growth of soybean roots and stems. After applying the bacterial fertilizer, the soil pH can be increased and the soil environment can be improved, so that the available potassium, alkaline nitrogen, available phosphorus and organic matter in the soil are all improved. The bacterial fertilizer can not only reduce the toxicity of aluminum to the soil, but also promote the growth of plants and increase soil fertility.

[0122] The present invention utilizes the characteristics of fast and unlimited reproduction of microorganisms to reduce the number of applications and has no secondary damage to the soil. It has the advantages of low cost, easy operation, resource conservation, and environmental protection. It provides a new direction for the improvement of aluminum-toxic soil and has certain research significance.

[0123] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein, but these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A strain of Pantoea agglomerans ALC, characterized in that: It was deposited in the General Microbiology Center of China Microorganism Culture Collection Administration on July 15, 2024, with the deposit number: CGMCC No.31309.

2. Use of a strain of Pantoea agglomerans ALC as claimed in claim 1 in preparing a bacterial fertilizer for promoting plant growth in aluminum-toxic soil.

3. The use according to claim 2, characterized in that: The functions of the Pantoea agglomerans ALC are: (1) Phosphate solubilization; (2) potassium release; (3) Nitrogen fixation; (4) Producing citric acid; (5)Production of IAA; (6) Producing iron carriers.

4. Aluminum-toxic soil plant growth-promoting bacterial fertilizer, characterized in that: It comprises a strain of Pantoea agglomerans ALC as described in claim 1.

5. The aluminum-toxic soil plant growth-promoting bacterial fertilizer according to claim 4, characterized in that: Also includes fulvic acid.

6. The aluminum-toxic soil plant growth-promoting bacterial fertilizer according to claim 5, characterized in that: After Pantoea agglomerans ALC was fermented and cultured, fulvic acid and the fermentation broth of Pantoea agglomerans ALC were mixed evenly according to the OD600 value of the fermentation broth of Pantoea agglomerans ALC and the colony count of the OD600 value so that the effective viable bacteria count in the fulvic acid was greater than 2.0×10 8 cfu / g.

7. The method for preparing the aluminum-toxic soil plant growth-promoting bacterial fertilizer according to claim 4, characterized in that: The following steps are involved: Pantoea agglomerans ALC was inoculated into LB liquid medium for fermentation culture; the fermentation liquid of Pantoea agglomerans ALC was mixed with fulvic acid evenly, and the effective viable bacteria count of fulvic acid was adjusted to >2.0×10 8 cfu / g, prepared into solid bacterial fertilizer.

8. The preparation method according to claim 7, characterized in that: The fermentation culture conditions are: 30° C., 180 rpm, culturing for 2 days.

9. The preparation method according to claim 7, characterized in that: The LB liquid culture medium contains 5 g / L yeast extract, 10 g / L tryptone, 10 g / L sodium chloride, and a pH of 4.5; the LB solid culture medium contains 5 g / L yeast extract, 10 g / L tryptone, 10 g / L sodium chloride, and 40 g / L agar, and a pH of 4.

5.

10. Use of the aluminum-toxic soil plant growth-promoting bacterial fertilizer as claimed in claim 4 in aluminum-toxic soil remediation and plant growth promotion.

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